Project Summary/Abstract The long-term objectives of this proposal include the following: 1) to determine the frequency and necessity of pioneer factor-nucleosome interactions genome wide during cell differentiation, and 2) to characterize the individual and cooperative pioneer factor-nucleosome interactions that occur during induced pluripotency. Cell fate decisions require exquisite spatiotemporal regulation of transcription factor (TF) binding to enhancers to mediate gene regulation, which is frequently co-opted in a variety of diseases. It is well established that active enhancers are locally depleted of nucleosomes, and that TF binding is coincident with, and often required for, local chromatin accessibility. However, it remains unclear how TFs might play an active role in displacing nucleosomes at enhancers. One hypothesis is that ?pioneer? TFs, so called because they are expressed early in cell differentiation and bind to their enhancer targets before they are rendered strongly accessible, directly bind and displace nucleosomes to facilitate accessibility. However, such a mechanism has never been validated on individual templates in vivo, chiefly due to the difficulty in testing for the presence of the putative intermediate TF-nucleosome particle. Lacking a direct test for in vivo pioneer activity, putative mechanisms for nucleosome eviction by pioneer TFs remain untested, and the locus specificity of pioneer activity is largely unknown. Moreover, the frequency and importance of TF pioneer activity in either natural differentiation systems or TF overexpression contexts during cellular reprogramming remain uncharacterized. To directly test the pioneer TF model, I will leverage an in situ, micrococcal nuclease (MNase) digestion-based alternative to ChIP-seq known as CUT&RUN, in which DNA fragment size is directly informative of the minimal protein-protected sequence, and therefore can distinguish direct TF binding from binding through a nucleosomal intermediate. I will use this information to determine the genome- wide landscape of pioneer factor-nucleosome interactions in embryonic stem cell differentiation and during induced pluripotency, and test the necessity of such interactions using functional genetic experiments. These insights will help us to understand how pioneer factors function to activate pathogenic gene expression in diseases such as cancer. I feel that my proposed project fits the mission of NIGMS to fund research in fundamental biology that uncovers new insights that could positively impact human health. !